Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is an unbranched chain of up to several thousand .alpha.-D-glucopyranose units linked by .alpha.-1-4 glycosidic bonds. Amylopectin is a highly branched molecule made of up to 50,000 .alpha.-D-glucopyranose residues linked by .alpha.-1-4 and .alpha.-1-6 glycosidic bonds. Approximately 5% of the glycosidic linkages in amylopectin are .alpha.-1-6 bonds, which leads to the branched structure of the polymer.
Amylose and amylopectin molecules are organized into granules that are stored in plastids. The starch granules produced by most plants are 15-30% amylose and 70-85% amylopectin. The ratio of amylose to amylopectin and the degree of branching of amylopectin affects the physical and functional properties of the starch. Functional properties, such as viscosity and stability of a gelatinized starch, determine the usefulness and hence the value of starches in food and industrial applications. Where a specific functional property is needed, starches obtained from various crops such as maize, rice, or potatoes may meet the functionality requirements. If a starch does not meet a required functional property, if for example it must have stable viscosity under high temperatures and acidic conditions, the functionality can sometimes be achieved by chemically modifying the starch. Various types and degrees of chemical modification are used in the starch industry, and the labelling and use of chemically modified starches must meet government regulations.
Within the starch bearing organs of plants, the proportion of amylose to amylopectin and the degree of branching of amylopectin are under genetic control. For example, plants homozygous recessive for the waxy gene lack a granule bound starch synthase enzyme and produce nearly 100% amylopectin. Plants homozygous recessive for the amylose extender gene can produce starch granules that are up to 90% amylose. The dull gene has been shown to control the activity of a starch branching enzyme.
Genes that have their primary effect on starch or protein synthesis, including amylose extender (ae), brittle (bt), dull (du), floury (fl), horny (h), opaque (o), shrunken (sh), sugary (su), and waxy (wx), are referred to as recessive genes because their effect on kernel appearance can be masked in F1 seed by the presence of a dominant form of each respective gene. Conventional nomenclature of plant varieties has been established to identify genotypes that carry a particular gene of interest. For the previously listed genes, genotypes are identified by the homozygous recessive mutant alleles they carry. Other genes not listed in the variety name are homozygous dominant. For example, a variety described as ae wx is homozygous recessive for the amylose extender and waxy genes and homozygous dominant for the other starch biosynthesis genes such as brittle, dull, shrunken, and sugary. According to conventional genetics teaching, the effect of a recessive gene is not expressed unless the gene is homozygous recessive. Hence, reports on the properties of starch from mutant plant species typically describe starch obtained from plants homozygous recessive for a particular gene or combination of genes. The properties of starch obtained from maize plants homozygous recessive for ae, du, wx, and aewx are reported in an article by E. Brockett et al. entitled "Gelatinization Characteristics of Starch from du, wx, ae, and aewx Endosperm of Sweet Corn Inbred Ia5125", published in Starch/Starke 40 (1988) Nr. 5. pp. 175-177.
In cereal grains such as maize (Zea mays L.), the kernel is the product of double fertilization (Kiesselbach, T. A., 1980, The Structure and Reproduction of Corn, University of Nebraska Press). The pollen grain contains two sperm nuclei. At the time of fertilization one of the sperm nuclei fuses with the nucleus of the ovule to form the embryo of the seed, and one fuses with two female nuclei to form the endosperm of the seed. The endosperm receives two thirds of its genetic material from the female plant and one third from the pollen. The immediate effect of pollen on the developing seed is termed xenia. The number of copies of a particular gene present in a cell, such as an endosperm cell, is known as the gene dose. Gene dosage effects have been studied for the ae and wx genes. In non-waxy maize, the ae allele is usually completely recessive to the dominant allele Ae with respect to kernel appearance. In waxy maize, kernels with varying doses of the ae allele can often be visually distinguished from each other. The effect on starch properties of various doses of ae in waxy maize has been shown by T. Yamada et al. in an article entitled "A Novel Type of Corn Starch from a Strain of Maize" published in Starke 30 (1978) Nr. 5, pp. 145-148. The interaction of various doses of ae and wx on starch accumulation and apparent amylose content was reported by Boyer et al. in The Journal of Heredity, 67:209-214 1976. No reports of the effect of various doses of du, su1, or sh1 on the properties of starch have been presented in the art.
Several U.S. patents describe the utility of various homozygous recessive gene combinations on starch production. The patents describe starch extracted from plants homozygous recessive for double or triple mutant gene combinations. For example, U.S. Pat. No. 4,789,557 relates to starch extracted from a plant homozygous recessive for the du and wx genes and U.S. Pat. No. 5,009,911 relates to starch extracted from a plant homozygous recessive for the ae and wx genes. There have been no patents describing the use of starch obtained from heterozygous grain.
Most cereal crops are handled as commodities, and many of the industrial and animal feed requirements for these crops can be met by common varieties which are widely grown and produced in volume. However, there exists at present a growing market for crops with special end-use properties which are not met by grain of standard composition. Most commonly, specialty maize is differentiated from "normal" maize, also known as field corn, by altered endosperm properties, such as an overall change in the degree of starch branching as in waxy or high amylose maize, an increased accumulation of sugars as in sweet corn, or an alteration in the degree of endosperm hardness as in food grade maize or popcorn; Glover, D. V. and E. T. Mertz, 1987, Corn. In: Nutritional Quality of Cereal Grains; Genetic and Agronomic Improvement, R. A. Olson and K. J. Frey, eds. American Society of Agronomy, Madison, Wis., pp. 183-336; Rooney, L. W. and S. O. Serna-Saldivar, 1987, Food Uses of Whole Corn and Dry-Milled Fractions, In: Corn:Chemistry and Technology, S. A. Watson and P. E. Ramstead, eds. American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 399-429. "Specialty" crops are typically grown under contract for specific end users who place value on starch quality or other specific quality attributes. A specialty crop such as waxy maize is more valuable as a raw material to the starch industry than is normal or commodity grade maize, and thus is referred to as a value added crop. Currently the market size and added value of waxy maize is such that approximately 150,000 acres are grown in the United States. Farmers are paid a premium for growing specialty crops such as waxy maize because it is more valuable than normal maize and must not be mixed with normal maize. The current invention offers the buyers of value added crops like waxy maize a source of starch having properties superior to waxy starch and offers farmers the opportunity to grow a higher value crop than normal or waxy maize.
Purified starch is obtained from plants by a milling process. Maize starch is extracted from kernels through the use of a wet milling process. Wet milling is a multi-step process involving steeping and grinding of the kernels and separation of the starch, protein, oil, and fiber fractions. A review of the maize wet milling process is given by S. R. Eckhoff in the Proceedings of the Fourth Corn Utilization Conference, Jun. 24-26, 1992, St. Louis, MO., printed by the National Corn Growers Association, CIBA-GEIGY Seed Division and the United States Department of Agriculture. Starch is used in numerous food and industrial applications and is the major source of carbohydrates in the human diet. Typically, starch is mixed with water and cooked to form a thickened gel. Three important properties of a starch are the temperature at which it cooks, the viscosity the gel reaches, and the stability of the gel viscosity over time. The physical properties of unmodified starch during heating and cooling limit its usefulness in many applications. As a result, considerable effort and cost is needed to chemically modify starch in order to overcome these limitations of starch and to expand the usefulness of starch in industrial applications.
Some limitations of unmodified starches and properties of modified starches are given in Modified Starches: Properties and Uses, O. B. Wurzburg, ed., 1986, CRC Press, Inc., Boca Raton, FL. Unmodified starches have very limited use in food products because the granules swell and rupture easily, thus forming weak bodied, undesirable gels. Depending on the food or industrial application, shortcomings of unmodified starches include excess or uncontrolled viscosity after cooking; cohesive or rubbery texture of cooked starch; structural break down during cooking or when exposed to shear or to low pH; and lack of clarity and the tendency of starch to become opaque and gel when cooled. Chemical modifications are used to stabilize starch granules thereby making the starch suitable for thousands of food and industrial applications including baby foods, powdered coffee creamer, surgical dusting powders, paper and yarn sizings, and adhesives. Common chemical modifications include cross linking in which chemical bonds are introduced to act as stabilizing bridges between starch molecules, and substitution in which substituent groups such as hydroxyethyl, hydroxypropyl or acetyl groups are introduced into the starch molecules.
Cross linking and substitution are multi-step processes involving reactions that are usually run on aqueous suspensions of starch at wide ranges of temperature and pH. Cross linking reactions are often run for 1 to 5 hours at 40.degree. C. to 50.degree. C. and pH 8 to 12. However, cross linking under acidic conditions and for up to 28 hours is necessary for some applications; Wurzburg, O. B., 1986, Cross-Linked Starches, In Modified Starches: Properties and Uses; O. B Wurzburg, ed.; pp. 41-53. Cross linking reinforces hydrogen bonds in starch granules with chemical bonds between molecules. When aqueous suspensions of non-cross linked starches are heated, hydrogen bonds weaken, allowing water to enter the granules, causing them to swell, fragment, rupture, and collapse. When this happens, the starch develops a cohesive, rubbery texture. Cross linking reinforces the hydrogen bonds upon heating, thus providing varying degrees of granule stability, depending on the number of cross links. Cross linked starches are used to a wide extent in foods, paper, textiles, and adhesives. Other chemical modifications, such as substitutions, very often depend on cross linking to impart a desired property.
Cross linked starches are used in foods, textiles, and adhesives, with the main use for high viscosity starches being as thickeners for food products; Jarowenko, W., 1986, Acetylated Starch and Miscellaneous Organic Esters. In Modified Starches: Properties and Uses, O. B. Wurzburg, ed., CRC Press, Boca Raton, Fla., pp. 55-77. Food starch thickeners must be stable under various conditions such as low pH, high speed mixing (shear), refrigeration, and freeze-thaw cycles. Cross linking provides resistance to low pH and shear, but the starches synerese (lose water holding capacity) during refrigeration. Therefore, cross linking is often combined with substitution to improve the thickening performance of starch. Cross-linked starches are stabilized by the addition of substituents such as acetyl, phosphoryl, and hydroxypropyl groups. The substitution reactions normally require the use of high concentrations of a salt to prevent starch granule gelatinization under the required conditions of high temperature and high pH. These cross-linked, substituted starches are used in baked, frozen, canned, and dry foods. Common uses are in pie fillings, gravies, custards, and cream fillings.
The use of chemically modified starches in the United States is regulated by the Food and Drug Administration (FDA). The Federal Food, Drug, and Cosmetic Act allows for two types of modified starches to be used in the food industry, "food starch-modified" and "industrial starch-modified". Food starch-modified may be used in food but must meet certain treatment limits, and industrial starch-modified may be used in items such as containers that come in contact with food and must also meet specified treatment requirements; Code of Federal Regulations, Title 21, Chapter 1, Part 172, Food Additives Permitted in Food for Human Consumption, Section 172, 892, Food Starch-Modified, U.S. Government Printing Office, Washington, D.C. 1981; (a) Part 178, Indirect Food Additives, Sect. 178.3520, Industrial Starch-Modified. These regulations limit the degree of chemical modification by defining the maximum amount of chemical reagent that can be used in the modification steps. The levels of by-products in starch resulting from the modification process are also regulated. For example, propylene chlorohydrin residues in hydroxypropyl starch are of special concern; Tuschhoff, J. V., 1986, Hydroxypropylated Starches, In Modified Starches: Properties and Uses, O. B. Wurzburg, ed., CRC Press, Boca Raton, FL, pp. 55-77.
The present invention offers a way to produce starch with the functional properties of certain chemically modified starches, without the need for chemical modification. Starch of the present invention can be used in food products as a direct replacement for chemically modified starch. By eliminating the chemical modification steps, the present invention offers the starch industry considerable cost and time savings, and dramatic reductions in waste treatment needs. Also, the present invention offers the starch industry a new type of starch with physical and functional properties not attainable through chemical modification. This new starch is also expected to serve as a superior base starch for chemical modifications by providing superior functionality at much lower levels of cross-linking and substitution than are currently required using a normal waxy base starch.